Genetic Variation of the Toll-Like Receptors in a Swedish Allergic Rhinitis Case Population

http://www.diva-portal.org This is the published version of a paper published in BMC Medical Genetics. Citation for the original published paper (version of record): Henmyr, V., Carlberg, D., Manderstedt, E., Lind-Halldén, C., Säll, T. et al. (2017) Genetic variation of the toll-like receptors in a Swedish allergic rhinitis case population. BMC Medical Genetics, 18(1): 18 https://doi.org/10.1186/s12881-017-0379-6 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:hkr:diva-16592 Henmyr et al. BMC Medical Genetics (2017) 18:18 DOI 10.1186/s12881-017-0379-6 RESEARCHARTICLE Open Access Genetic variation of the Toll-like receptors in a Swedish allergic rhinitis case population V. Henmyr1,2*†, D. Carlberg2†, E. Manderstedt1,2, C. Lind-Halldén2, T. Säll1, L. O. Cardell3 and C. Halldén2 Abstract Background: Variation in the 10 toll-like receptor (TLR) genes has been significantly associated with allergic rhinitis (AR) in several candidate gene studies and three large genome-wide association studies. These have all investigated common variants, but no investigations for rare variants (MAF ≤ 1%) have been made in AR. The present study aims to describe the genetic variation of the promoter and coding sequences of the 10 TLR genes in 288 AR patients. Methods: Sanger sequencing and Ion Torrent next-generation sequencing was used to identify polymorphisms in a Swedish AR population and these were subsequently compared and evaluated using 1000Genomes and Exome Aggregation Consortium (ExAC) data. Results: The overall level of genetic variation was clearly different among the 10 TLR genes. The TLR10-TLR1-TLR6 locus was the most variable, while the TLR7-TLR8 locus was consistently showing a much lower level of variation. The AR patients had a total of 37 promoter polymorphisms with 14 rare (MAF ≤ 1%) and 14 AR-specific polymorphisms. These numbers were highly similar when comparing the AR and the European part of the 1000Genomes populations, with the exception of TLR10 where a significant (P = 0.00009) accumulation of polymorphisms were identified. The coding sequences had a total of 119 polymorphisms, 68 were rare and 43 were not present in the European part of the 1000Genomes population. Comparing the numbers of rare and AR-specific SNPs in the patients with the European part of the 1000Genomes population it was seen that the numbers were quite similar both for individual genes and for the sum of all 10 genes. However, TLR1, TLR5, TLR7 and TLR9 showed a significant excess of rare variants in the AR population when compared to the non-Finnish European part of ExAC. In particular the TLR1 S324* nonsense mutation was clearly overrepresented in the AR population. Conclusions: Most TLR genes showed a similar level of variation between AR patients and public databases, but a significant excess of rare variants in AR patients were detected in TLR1, TLR5, TLR7, TLR9 and TLR10.Thisfurther emphasizes the frequently reproduced TLR10-TLR1-TLR6 locus as being involved in the pathogenesis of allergic rhinitis. Keywords: Allergic rhinitis, Mutation spectrum, Next-generation sequencing, Rare variants, Toll-like receptor * Correspondence: [email protected] †Equal contributors 1Department of Biology, Lund University, Lund, Sweden 2Department of Biomedicine, Kristianstad University, 291 39 Kristianstad, Sweden Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Henmyr et al. BMC Medical Genetics (2017) 18:18 Page 2 of 10 Background Methods Allergic rhinitis (AR) is a global illness with a well- The present study aims to describe the genetic variation recognized impact on quality of life and work performance. of the promoter and coding sequences of the 10 TLR It is characterized by nasal obstruction, secretion and genes in a Swedish AR population of 288 patients. As itching and is often associated with symptoms of the eyes, the first larger resequencing effort in AR, this could fatigue and asthma [1]. The heritability of AR has been esti- identify rare variants that are increasing risk for AR and mated to 0.66–0.78 [2, 3] and its development is generally identify potential targets for future studies. believed to be the result of an interaction between genetical and environmental factors. Toll-like receptors (TLRs) are a Study samples well-established group of pattern-recognition receptors The 288 AR patients (140 females, 148 males, mean age encompassing ten members which initiate innate immune 33 years) were recruited at Malmö University Hospital responses [4]. Contrary to this, TLR10 has recently been (Malmö, Sweden) between the years 2003 and 2009 and shown to have inhibitory effects by down-regulating TLR2 consist of unrelated individuals from the general popula- mediated immune response [5]. The TLRs are an important tion. All patients were of Caucasian origin, with both barrier between environment and the organism and have parents born in Sweden. They were patients at the been suggested to play a key role in the development of allergy clinic and were diagnosed with symptomatic allergic disease in growing infants [6]. birch and/or timothy grass pollen induced intermittent The 10 TLR genes are located on five different chromo- AR (for more details see Additional file 1a). As described somes. TLR1, TLR6 and TLR10 arelocatedina90kbp in Nilsson et al. 2012, diagnostic procedures for the region on chromosome 4, TLR7 and TLR8 are located in a study population included face-to-face personal inter- 57 kbp region on the X chromosome, whereas the view of medical history and skin prick tests (SPT) [22] remaining genes are located in separate loci on chromo- or Phadiatop tests with at least a class two response to some 1 (TLR5), chromosome 3 (TLR9), chromosome 4 birch and/or timothy grass pollen. A total of 59% of the (TLR2 and TLR3) and chromosome 9 (TLR4). Earlier patients showed a positive SPT for both allergens. SPT studies of genetic variation have reported a large number were performed with a standard panel of 11 common of polymorphisms for all 10 TLR genes [7, 8]. More airborne allergens (ALK-Abelló, Hörsholm, Denmark) recently, the 1000Genomes project has re-sequenced (for more details see Additional file 1b). SPT were 1092 healthy individuals from 14 populations using performed on the volar side of the forearm with saline low-coverage whole-genome and high-coverage exome buffer as negative and histamine chloride (10 mg/ml) as sequencing. In concordance with previous studies, the positive controls. A wheal reaction diameter of ≥3mm 1000Genomes populations also show high levels of was considered a positive SPT response. Atopy is de- variation in the TLR genes [9]. fined as a positive SPT reaction to at least one of the The TLR genes have been significantly associated with tested allergens and AR is diagnosed based on the pres- various allergic phenotypes in linkage and candidate ence of atopic status and typical AR symptoms as gene studies [10–14]. Single nucleotide polymorphisms defined by the Allergic Rhinitis Impact on Asthma (SNP) in TLR7 and TLR8 have been found to be associ- (ARIA) guidelines. None of the patients suffered from ated with AR in two independent studies [15, 16]. Three severe asthma and less than 10% had moderate asthma meta-genome-wide association studies (GWAS) have an- with continuous medication. Genomic DNA was isolated alyzed various allergic phenotypes including self- from blood collected in EDTA using the QIAmp DNA reported AR and allergic sensitization [17–19]. A total Blood kit (Qiagen, Hilden, Germany) and DNA concen- of 37 loci were associated with allergic disease and four trations were determined by fluorometry using Pico- of these loci were reported by all three studies. The Green (Molecular Probes, Eugene, OR, USA). TLR10-TLR1-TLR6 locus was one of these 4 loci. Follow- ing these studies, a replication attempt of the SNPs iden- Sanger sequencing tified in the three meta-GWAS was performed using Primers were designed using NCBI Primer-BLAST both the original phenotype definitions and a more strict (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amp- AR definition [20]. A total of eight loci were successfully lify at least 50 bp downstream and 500 bp upstream of the replicated. The TLR10-TLR1-TLR6 locus was replicated start of exon 1 of all 10 TLR genes (for exact coordinates using three index SNPs and all phenotype definitions, and primer sequences see Additional files 2 and 3). Big including a more strictly defined AR phenotype. Also, an Dye Terminator Sanger sequencing was performed in additional meta-GWAS that analyzed a combined both directions using a 3130XLGenetic Analyzer (Applied phenotype of asthma and hay fever identified a total of Biosystems, Foster City, CA, USA). Sequences were inter- 10 loci, including the TLR10-TLR1-TLR6 locus, as asso- preted and all polymorphisms were identified using Seq- ciated with the disease [21]. Scape ver. 2.5 (Applied Biosystems) and confirmed by Henmyr et al. BMC Medical Genetics (2017) 18:18 Page 3 of 10 manual inspection. Further information about Sanger populations and the three statistics were summarized for sequencing can be found in Additional file 4.

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